Tumour-specific vector for gene therapy

ABSTRACT

The invention relates to a vector for the gene therapeutic treatment of tumours, especially in connection with radiotherapy. Said vector is provided with a therapeutic gene in the DNA sequence thereof. The gene is controlled by the promoter for the catalytic subunit of the telomerase or by the promoter for cyclin A.

RELATED APPLICATION

[0001] This is a continuation application of International PatentApplication PCT/EP00/08921, in which the United States is a designatedcountry.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a vector for treating tumours bygene therapy, in particular in connection with a radiotherapy, whose DNAsequence has at least one tissue-specific promoter and at least onetherapeutic gene whose expression is controlled by the promoter.

[0004] 2. Related Prior Art

[0005] DE 44 44 949 C1 discloses a vector of this type.

[0006] For the purposes of the present invention, “tumours” means bothmalignant and benign tumours.

[0007] Malignant neoplastic disorders account for approx. 30% of deathsin the civilized world, and there is at present no safe therapy for anytumour yet, in spite of worldwide efforts over the last decades. Manytumours can be treated only with difficulties, if at all.

[0008] Examples of benign tumours include tumours of the vascular wallfor which partly the same therapies are used as for the treatment ofmalignant tumours, i.e. cancer. Such tumours of the vascular wall formas recurring stenoses essentially due to induction of smooth muscle-cellproliferation in the vascular wall caused by “balloon dilatations”(PTCA) for the therapy of local stenoses of the vascular wall which maylimit an organ's blood supply. The treatment of such arterioscleroticdisorders includes in addition to balloon dilatation also bypasssurgery, stents and other alternative therapeutic methods which,however, likewise have the problem of recurring stenosis, i.e.constriction of the lumen of the treated vessel due to a benign tumour.

[0009] Besides surgical removal of both malignant and benign tumours andthe treatment thereof with cytostatics, radiotherapy represents from thepresent point of view one of the most important pillars of tumourtherapy. In this connection, the probability of destruction of thetumour depends on the dose administered, with the dose to beadministered being limited by the radiosensitivity of normal tissuewhich inevitably is also irradiated. Therapeutic successes thus dependon the relative radiosensitivity of the tumor cells compared with thecells of the neighboring tissue.

[0010] Consequently, an increase in the therapeutic range due toselective radiosensitization of tumour cells would mean a significantstep forward in the treatment of tumours and improve the rates of cure.For this reason, pharmacological radiosensitizers which ought to maketumour cells more sensitive to radiation are already in use clinically.

[0011] Gene therapy, for the first time, offers the opportunity ofachieving significant progress in controlling cancer by making itpossible to use therapeutic genes for enhancing radiotherapy or therapywith cytostatics.

[0012] In this connection, viral vector systems play a great part intransducing therapeutic genes. Besides retroviral vector systems which,to a certain extent, prefer proliferating cells and very often integrateinto the cellular genome, especially adenoviral vector systems whichmake it possible to attain a high titre of virus particles and whichhave good transduction efficiency and a very low rate of integration arein discussion; see Ali et al., Gene Ther. (1991), Volume 1, 367-384.

[0013] It is crucial for a reliable gene therapy to use the therapeuticgene only in the desired target cells. It would therefore be desirableto use in the gene therapy of tumours a tumour cell-specific promoterwhich is active in various tumour species and not active in all types ofnormal tissue. Such a promoter, however, has not yet been found.

[0014] In this connection, the initially mentioned DE 44 44 949 C1describes a vector for the gene therapy of patients who, after surgicalremoval of a tumour, undergo an aftertreatment using conventionalradiation and/or cytostatic methods. The vector contains an expressibleDNA insert which is located behind a promoter active in tumour cells andcodes for the DNA-binding domain (DBD) of a poly(ADP-ribose) polymerase(PARP). The idea on which that publication is based is to inhibit theactivity of the enzyme PARP which is required for repairing damaged DNAby adding DBD molecules so that repair of damaged DNA is prevented.

[0015] As example of a tissue-specific promoter the MVM P4 promoter ismentioned. The vector may be a viral vector, and the viruses arereplication-incompetent and can be complemented in trans in order toobtain viruses which code for DBD but are unable to propagate inpatients.

[0016] It has turned out to be a disadvantage of the known vector thatthe P4 promoter does not yet have the tissue specificity required for asafe gene therapy so that DNA repair is also inhibited in normal tissue,and this is, for reasons that need no further explanation, undesirable.

[0017] Most gene therapies therefore include an enhancement of thetumour-specific immune response, i.e. they are a priori systemic, sincethe local increase in the immune response is not limited to the tumour.At the same time, this is also a desired advantage, because in principleit is also possible to destroy metastases using this therapy.

[0018] In order to allow a more or less local gene therapy, the use ofretroviruses which transduce genes only into dividing cells has alreadybeen discussed. Furthermore, adenovirus mutants were proposed, whichought to replicate only in p53-negative tumour cells; Heise et al., Nat.Med. (1997), Volume 3, 639-645. It is assumed here that all p53-positivecells can prevent proliferation of the adenovirus.

[0019] In addition, Joki et al., Hum. Gene Ther. (1995), Volume 6,1507-1513 have already proposed using radiation-inducible promoters andNettelbeck et al., Adv. Exp. Med. Biol. (1998), Volume 451, 437-440proposed using cell cycle-specific promoters.

[0020] However, all of these known solutions have specificdisadvantages. The concept of immunotherapy has the principaldisadvantage that immunosuppressed patients cannot respond to it, theneoplastic disorder itself often additionally adversely affecting theimmune system of the said patients. Furthermore, it has turned out thatan immunotherapeutic gene or a “suicide gene” which kills the targetcell is on its own not sufficient for in vivo administrations; Uckert etal., HUM. GENE THER. (1998), Volume 9, 855-865. The combination ofseveral immunostimulatory genes, where appropriate in connection withsuicide genes, has also up to now not led to a breakthrough which wouldallow a standard application for a given neoplastic disorder.

[0021] The abovementioned retroviruses are furthermore not exclusivelyselective for tumours but infect all dividing cell types, as long as anappropriate receptor is present. Moreover, infection of a healthy cellcarries the risk of insertion mutagenesis, since retroviruses integrateinto the cellular genome. Replication-competent adenoviruses which havebeen proposed for a specific tumour therapy are also not specific fortumour cells, as has been proved since then, the effect also beingindependent of the p53 state; Rothmann et al., J. Virol. (1998), Volume72, 9470-9478.

[0022] The previously described use of suicide genes, too, hasdisadvantages, since the said suicide genes increase the side effects inhealthy proliferating tissues. This problem could be avoided only if thesuicide genes were expressed tumour-specifically.

SUMMARY OF THE INVENTION

[0023] In view of the above, it is an object of the present invention toprovide a vector of the type mentioned at the outset, which actstumour-specifically and has only slight side effects on normal tissue.

[0024] According to the invention, this object is achieved with thevector mentioned at the outset by the promoter being selected from thegroup comprising the promoter for the catalytic subunit of telomeraseand the promoter for cyclin-A.

[0025] The object on which the invention is based is completely achievedin this manner.

[0026] In fact, the inventors of the present application have recognizedthat the promoter for the catalytic subunit of telomerase isparticularly well suited for a tumour-specific gene therapy. Namely,this promoter which is described, for example, by Horikawa et al.,Cancer Res. (1999), Volume 59, 826-830, and Takakura et al., Cancer Res.(1999), Volume 59, 551-557 is very active in tumour cells and more than90% of all tumours are telomerase-positive. Furthermore, the saidpromoter is less active in a few proliferative stem cells, for examplegerm line cells and blood stem cells, and not active at all in the vastmajority of proliferative cell types such as, for example, endothelial,fibroblasts, hepatocytes, etc.

[0027] In this connection, WO 98/11207 discloses the use of a telomerasepromoter in connection with the cell transfection, in order to expressproducts which inhibit cell growth with regard to a cancer treatment.However, the said publication does not mention application of atelomerase promoter in connection with a radiotherapy.

[0028] The second promoter used according to the invention is thecyclin-A promoter which is activated in the S phase of the cell cycle,while being repressed in resting cells, i.e. in the G0 and early G1phases; Henglein et al., PNAS (1994), Volume 91, 5490-5494. Cyclin-A isinvolved in several regulatory pathways in cell division so that it isexceptionally well suited to expressing therapeutic genes inproliferating cells. Here, the inventors of the present applicationstart from the finding that tumour cells are distinguished in particularby defects in cell cycle regulation and, as a result, enter the S phasein an uncontrolled manner. Choosing the cyclin-A promoter thus wouldmake the therapeutic gene express only in the tumour but not in thesurrounding normal tissue, since this tissue in most cases has no oronly little proliferative activity.

[0029] The cyclin-A promoter in combination with a therapeutic gene isparticularly suitable for treating benign tumours, especially inconnection with the recurring stenoses mentioned at the beginning, whosetherapy is considerably improved by the vector of the invention. Againstthis background, the present invention also relates to the use of thecyclin-A promoter in connection with a therapy of benign tumours, inparticular in connection with recurring stenoses.

[0030] Besides the safety provided by the tumour-specific promoters forthe catalytic subunit of telomerase and for cyclin-A, a second point ofsafety results from applying these promoters or the therapeutic genescontrolled thereby in connection with a radiotherapy which, according tothe current state of the art, can be directed very locally only towardsthe tumour tissue.

[0031] By combining various therapeutic genes which are expressedtissue-specifically and the locally directed radiotherapy, it ispossible to provide for the tumour cells to react more sensitively thanthe neighbouring tissue, albeit perhaps only by a small factor, but as aresult the tumour can then be destroyed efficiently.

[0032] Besides using the new vector in radiotherapy, it is also anobject to use it in connection with chemotherapy, because in this way itis possible to destroy non-localized metastases.

[0033] In this connection, preference is given to the therapeutic genecoding for a protein selected from the following group of proteins:cytosine deaminase (CD), herpes simplex-virus thymidine kinase (HSV-TK),DNA-binding domain (DBD) of poly(ADP-ribose) polymerase (PARP),cytotoxic protease 2A and 3C of picornaviruses, preferably ofenteroviruses, more preferably of group B Coxsackie viruses (CVB), inparticular serotype B3.

[0034] Cytosine deaminase converts 5-fluorocytosine to 5-fluorouracilwhich is incorporated into the DNA of replicating cells and then killsthese cells. A systemic 5-fluorocytosine treatment in connection withlocal radiotherapy leads to a specific increase in the destruction oftumours, since cytosine deaminase is only formed in the tumour cells sothat the dreaded side effects such as necroses/fibroses in neighbouringtissue, damage of bone marrow and intestinal mucosa, etc. are avoided.

[0035] HSV-TK acts in a similar way; this enzyme activates gancyclovirwhich likewise incorporates into the DNA of replicating cells anddestroys the DNA so that, in connection with local radiotherapy, thesame advantages as with CD are attained.

[0036] In contrast to CD and HSV-TK, expression of DBD molecules leadsto inhibition of the activity of PARP which is required for repairingDNA damage. In this way it is not possible to “repair” again tumourcells “predamaged” in connection with the local radiotherapy, so thatthey die.

[0037] In contrast, the proteases 2A and 3C induce apoptosis in cellsand are thus cytotoxic.

[0038] The inventors of the present application have now found that bythe combination of firstly radiotherapy and secondly of proteins of theabove-mentioned type that are tissue-specifically expressed by the twoabove-mentioned promoters a selective destruction of tumours can beachieved, which is markedly more effective than the individual measuresused previously.

[0039] In this connection, preference is given to the therapeutic genebeing a fusion gene coding for a fusion protein of at least two proteinsselected from the abovementioned group of proteins, the therapeuticgene, preferably between the sequence regions for the two proteins,coding for a peptide linker which preferably comprises glycine, inparticular 8-10 glycines.

[0040] It is advantageous here that a synergistic effect can be achievedif the therapeutic gene contains two suicide genes, with differentmechanisms of action of the partners within the fusion proteins inparticular producing a synergistic action. The advantage of expressingfusion genes is the possibility of transferring simultaneously twodifferent therapeutic principles and thus producing additive orfrequently even synergistic effects in the therapy. In order for theprotein domains of the fusion partners to be able to fold optimally forthe application, it may be sensible to clone the information for a shortpeptide linker, preferably 8-10 glycines, between the cDNAs.

[0041] Particular preference is given to the following fusion proteins:

[0042] CD-linker-HSV-TK, CD-linker-DBD, CD-linker-2A, CD-linker-3C,HSV-TK-linker-DBD, HSV-TK-linker-2A, HSV-TK-linker-3C, DBD-linker-2A andDBD-linker-3C. The order of the fusion partners within a fusion proteinmay also be reversed.

[0043] Overall, preference is given to the vector being based on a virusvector, in particular on an adenovirus vector or an adeno-associatedvirus vector (AAV).

[0044] The invention has as a further object a retrovirus vector codingfor the novel vector.

[0045] Thus, the novel gene therapy system may be introduced both viaconventional viral or non-viral vectors and also via a retrovirus vectorinto the organism in which it has a substantially more selective effecton the tumour than has previously been possible.

[0046] Furthermore, preference is given to providing, between thepromoter and the therapeutic gene, a positive-feedback system which isdriven by the promoter and controls by itself the expression of thetherapeutic gene, the positive-feedback system preferably comprising theT7 promoter and the gene for T7 RNA polymerase, and preference isfurthermore given to the promoter controlling the gene for T7 RNApolymerase and the T7 promoter controlling the therapeutic gene, withfurthermore a second expression unit being provided, which contains theT7 RNA polymerase under the control of the T7 promoter.

[0047] This system of positive feedback causes increased expression ofthe therapeutic gene in the target cells, with the promoter, i.e. thetelomerase or cyclin-A promoter making sure that the positive-feedbacksystem is “triggered” only in the target cells. This positive feedbackis based on the finding that the T7 promoter is silent in eukaryoticcells without said T7 RNA polymerase, thus providing a very safe systemwhich multiplies the therapeutic effect without displaying side effects.

[0048] In a first expression unit the telomerase promoter, for example,controls T7 RNA-polymerase expression. The second expression unit thencontains the T7 promoter which again controls T7 RNA-polymeraseexpression. In this way, production of T7 RNA polymerase is increasedvia positive feedback. Since in the third expression unit thetherapeutic gene is under the control of the T7 promoter, T7RNA-polymerase expression by positive feedback also increases expressionof the therapeutic gene.

[0049] It is understood that the features mentioned above and thosestill to be illustrated below can be used not only in the combinationsindicated in each case but also in other combinations or on their own,without leaving the context of the present invention.

[0050] Further features and advantages of the invention arise from thefollowing description of preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The examples below are illustrated on the basis of the attacheddrawing in which:

[0052]FIG. 1 shows viral vectors for expressing therapeutic genes intumour cells; and

[0053]FIG. 2 shows viral vectors with positive-feedback expression oftherapeutic genes in tumour cells.

DESCRIPTION OF PREFERRED EMBODIMENTS Example 1

[0054] Generation of the Building Blocks for the Viral Vectors

[0055] 1.1 Telomerase promoter: The telomerase promoter sequence isknown; Horikawa et al., loc. cit. and Takakura et al., loc. cit. Thepromoter may be amplified from cells (e.g. HeLa tumour cells) via PCRusing, for example, the following primers:

[0056] Forward primer: ATC AGC TTT TCA AAG ACA CAC

[0057] Reverse primer: CGC GGG GGT GGC CGG GGC CAG

[0058] 1.2 Cyclin-A promoter: The cyclin-A promoter sequence islike-wise known; Henglein et al., loc. cit. The promoter may beamplified from cells, for example HeLa tumour cells, via PCR using thefollowing primers:

[0059] Forward primer: CGT GTT AAA TAA TTT ATG CAC

[0060] Reverse primer: CAC TGC TCC CGG GAG TGG ACG

[0061] 1.3 T7 promoter: The T7 RNA-polymerase promoter (approx. 30 bp)may be obtained from plasmid pCR-Script (Stratagene) by BssHI/KpnIdigest. The T7-promoter and T7 RNA-polymerase sequences are described,for example, in Dunn and Studier, J. Mol. Biol. (1983), Volume 166,477-535; GenBank Accession No. V01146, J02518, X00411.

[0062] 1.4 Cytosine deaminase (CD): The CD gene (1.3 kb) may be obtainedfrom plasmid pcDNA3-CD of the applicant by BamHI/NotI digest. The CDsequence has been described, for example, by Austin and Huber, Mol.Pharmacol. (1993), Volume 43, 380-387; GenBank Accession No. S56903.

[0063] 1.5 Herpes simplex virus thymidine kinase (HSV-TK): The HSV-TKgene (1.1 kb) can be obtained from plasmid pCDTK of the applicant byBamHI/BglI digest. The HSV-TK sequence is de-scribed by Suzutani et al.in Microbiol. Immunol. (1995), Volume 39, 787-794; GenBank Accession No.AB009255.

[0064] 1.6 DNA-binding domain (DPD) of poly(ADP-ribose) polymerase(PARP): The DBD (1.1 kb) may be obtained from plasmid pPARP6 byXbaI/SalI digest; Küpper et al., J. Biol. Chem. (1990), Volume 265,18721-18724.

[0065] 1.7 CVB3 protease 2A: The sequence coding for the cytotoxicpro-tease 2A is obtained from plasmid pIND-2A of the applicant by Pmedigest.

[0066] 1.8 CVB3 protease 3C: The sequence coding for the cytotoxicprotease 3C is obtained from plasmid pIND-3C of the applicant by Pmedigest.

[0067] Klump et al. describe the complete CVB3-cDNA sequence in J.Virol. (1990), Volume 64, 1573-1583; GenBank Accession No. M33854;protein 2A: Nucleotide 3304-3744; protein 3C: Nucleotide 5362-5910.

[0068] 1.9 Fusion genes: A fusion gene means the continuous sequence ofa therapeutic gene composed of several, preferably two, cDNAs which areexpressed via a single promoter to give a continuous fusion protein. Inthis way it is possible to transfer simultaneously two therapeuticprinciples, thus resulting in additive or synergistic effects in thetherapy.

[0069] In order for the protein domains of the fusion partners to beable to fold optimally for application, the information for a shortpeptide linker, preferably for glycine, in particular 8-10 glycines, iscloned between the cDNAs.

[0070] In this way, the following fusion genes are generated:

[0071] CD-linker-HSV-TK, CD-linker-DBD, CD-linker-2A, CD-linker-3C,HSV-TK-linker-DBD, HSV-TK-linker-2A, HSV-TK-linker-3C, DBD-linker-2A andDBD-linker-3C. The order of the fusion partners within a fusion gene mayalso be reversed.

[0072] 1.10 T7 RNA polymerase (T7 Pol): The T7 Pol cDNA (2.6 kb)originates from plasmid pAR3132 of the applicant and contains codons11-883 of the T7 RNA-polymerase gene.

[0073] The sequences of the building blocks have been deposited with thePubMed gene bank of the National Library of Medicine(http://ww4.ncbi.nlm.nih.gov/Pub-Med/).

Example 2

[0074] Preparation of Recombinant Adenoviruses

[0075] Recombinant adenoviruses are prepared by using, for example, theE1/E3-deleted adenovirus-5 system of Vogelstein; He et al., PNAS (1998),Volume 95, 2509-2514.

[0076] The cDNA to be expressed, i.e. the therapeutic gene, is clonedinto vector pShuttle (6.7 kb). The promoter (telomerase or cyclin-A)intended for application is cloned in front of the cDNA and apolyadenylation signal is cloned behind the cDNA. The newly generatedplasmid is transformed together with the pAdEasy1 helper plasmid intothe recombination-competent bacterial strain BJ5183.

[0077] Homologous recombination of the overlapping shuttle- andhelper-vector sequences results in a recombinant adenoviral genome whichis isolated from the bacteria and transformed into the recA-strain HB101for preparative processing. Plasmid material is then isolated from thesaid bacteria on the preparative scale and purified via caesium chloridecentrifugation.

[0078] The plasmid material obtained is transfected into theE1-expressing helper cell line 911; Fallaux et al., Hum. Gene Ther.(1996), Volume 7, 215-222. After transfection with the recombinantadenoviral genome, the cells are overlaid with soft agar. The virus thenpropagates in transfected cells and a plaque is formed from which therecombinant viruses can be isolated by freeze-thaw lysis. Expression canbe detected after two days but no cytopathic effect (CPE) is apparentyet. Virus stocks are obtained by infecting new 911 cells with theappropriate recombinant adenoviruses. After approx. four days, the CPEhas fully formed. The cells are disrupted in a Dounce homogenizer, celldebris is pelleted by short centrifugation and the viruses present inthe supernatant are purified via caesium chloride density-gradientcentrifugation.

[0079] The adenovirus vector obtained in this way is depicted in FIG. 1,top, and the abbreviations used are explained in the legend to thefigures.

Example 3

[0080] Preparation of Recombinant Adeno-associated Viruses (AAV)

[0081] An example of the system used here is the system of Samulski,comprising two plasmids plus adenovirus; Snyder et al.: “Production ofrecombinant adeno-associated viral vectors”. Current Protocols in HumanGenetics. New York: John Wiley and Sons (1996), 12.1.1-12.1.24.

[0082] The cDNA to be expressed, i.e. the therapeutic gene, is clonedtogether with regulatory sequences (promoter and poly-A signal) into avector containing only AAV-2 terminal repeats. These repeats are theminimum Cis-regulatory sequences required for replication and packaging;Xiao et al., J. Virol. (1997), Volume 71, 941-948.

[0083] The vector is generated by excising the rep/cap sequence via XbaIdigest from plasmid pSub201 (Human Gene Therapy Center, University ofNorth Carolina, Chapel Hill, N.C., USA). The terminal repeats of in eachcase 0.18 kb remain in the vector.

[0084] The building blocks intended for the particular gene transfersystem, such as promoter, cDNA of the therapeutic gene andpolyadenylation signal, are then cloned into said vector. The vectorplasmids thus generated are transfected into 293-cells, in each casetogether with the pAAV/Ad (Human Gene Therapy Center) helper plasmidwhich provides the AAV structural and non-structural proteins (cap andrep) in trans. On the next day, wild-type adenovirus is added as ahelper for AAV replication to the cells at an MOI of 3. Two to threedays after transfection/infection, the cytopathic effect (CPE) is wellvisible and recombinant AAV can be obtained from the cells.

[0085] The caesium chloride purification method is carried out asdescribed by Snyder et al., loc. cit. Essential elements of this AAVpurification are three times freeze-thaw lysis of the infected cellsplus ultrasound treatment to release the viruses, ammonium sulphateprecipitation to remove cellular proteins, purification of the AAVparticles on a CsCl gradient by ultracentrifugation, dialysis of thepurified AAV fractions by PBS and heat-inactivation of contaminatingadenoviruses by incubation at 56° C. for 15 minutes (AAV is notinactivated by this treatment).

[0086] The AAV vector obtained in this way is depicted in FIG. 1,centre.

Example 4

[0087] Preparation of Recombinant Retroviruses

[0088] An example of a system which may be used here is the system fromClonetech (Heidelberg). This system comprises shuttle vectors, forexample pLNCX (6.2 kb), which can be propagated via transformation intobacteria and also a helper cell line, the RetroPack PT67 line, whichenables transcomplementation of the vectors to virions.

[0089] Prior to using the pLNCX shuttle vector, the CMV promoter isremoved from this vector in order to be replaced thereafter by thepromoter of choice, i.e. the telomerase promoter or the cyclin-Apromoter. The abovementioned therapeutic genes or fusion genes are thencloned into the multiple cloning sequence with polyadenylation signals.

[0090] The recombinant vector is transfected into the packaging cellline PT67. Transfected cells can be selected for by using the antibioticG418. As a result, recombinant retroviruses are produced, which areobtained from cells and cell culture supernatant via methods analogousto those described in Examples 2 and 3 and which can be purified bycaesium chloride density-gradient centrifugation.

[0091] The retrovirus vector obtained in this way is depicted inprinciple in FIG. 1, bottom.

Example 5

[0092] Adenovirus Vector with Positive-feedback System

[0093]FIG. 2 depicts an adenovirus vector which was prepared asde-scribed in Example 2. However, instead of a single promoter and asingle therapeutic gene, this vector contains three expression units,the first of which contains T7 RNA polymerase under the control of thetelomerase promoter. The second expression unit likewise contains T7 RNApolymerase but under the control of its own promoter, the T7 promoter.Finally, the third expression unit contains the therapeutic gene underthe control of the T7 promoter.

[0094] When target cells are infected, first the telomerase promotercauses expression of T7 RNA polymerase (first expression unit). The T7RNA polymerase generated “switches on” the T7 promoter in the secondexpression unit, so that this T7 promoter, too, causes T7 RNA-polymeraseproduction. This results in a positive-feedback system, and the more T7RNA polymerase is generated, the more T7 promoter is switched on.

[0095] In this way, expression of the therapeutic gene which here isunder the control of a T7 promoter is increased.

[0096] In this system, the telomerase promoter thus controls expressionof the therapeutic gene not directly, as in Examples 2-4, butin-directly via the intermediate step of the positive-feedback sys-temof T7 promoter and T7 RNA polymerase.

[0097] Since the T7 promoter is silent in eukaryotic cells without theT7 RNA polymerase which usually is not found there, the therapeutic geneconsequently can be expressed only in those cells in which thetelomerase promoter is active, i.e. especially in tumour cells.

[0098] Therefore, what I claim, is:

1 4 1 21 DNA Artificial Sequence Forward primer for telomerase promoter1 atcagctttt caaagacaca c 21 2 21 DNA Artificial Sequence Reverse primerfor telomerase promoter 2 cgcgggggtg gccggggcca g 21 3 21 DNA ArtificialSequence Forward primer for Cyclin-A promoter 3 cgtgttaaat aatttatgca c21 4 21 DNA Artificial Sequence Reverse primer for Cyclin-A promoter 4cactgctccc gggagtggac g 21

What is claimed is:
 1. A vector for treating tumours by gene therapy,comprising a DNA sequence comprising a tissue-specific promoter and atleast one therapeutic gene under expression control by said promoter,wherein said promoter is selected from the group consisting of thepromoter for the catalytic subunit of telomerase and the promoter forcyclin-A.
 2. The vector of claim 1, wherein the therapeutic gene codesfor a protein selected from the group of proteins consisting of:cytosine deaminase (CD), herpes simplex-virus thymidine kinase (HSV-TK),DNA-binding domain (DBD) of poly(ADP-ribose) polymerase (PARP),cytotoxic protease 2A and 3C.
 3. The vector of claim 2, wherein saidcytotoxic protease 2A and 3C is from picornaviruses.
 4. The vector ofclaim 2, wherein said cytotoxic protease 2A and 3C is from group BCoxsackie viruses.
 5. The vector of claim 2, wherein said cytotoxicprotease 2A and 3C is from group B Coxsackie viruses, serotype B3. 6.The vector of claim 2, wherein the therapeutic gene is a fusion genecoding for a fusion protein of at least two proteins selected from thegroup of proteins.
 7. The vector of claim 6, wherein the therapeuticgene between the sequence regions for the two proteins codes for apeptide linker.
 8. The vector of claim 7, wherein the linker comprisesglycine.
 9. The vector of claim 8, wherein the linker comprises between8-10 glycines.
 10. The vector of claim 7, wherein the fusion gene isselected from the group consisting of: CD-linker-HSV-TK, CD-linker-DBD,CD-linker-2A, CD-linker-3C, HSV-TK-linker-DBD, HSV-TK-linker-2A,HSV-TK-linker-3C, DBD-linker-2A and DBD-linker-3C.
 11. The vector ofclaim 1, comprising a virus vector.
 12. The vector of claim 1,comprising an adenovirus vector.
 13. The vector of claim 1, comprisingan adeno-associated virus (AAV) vector.
 14. The vector of claim 1,wherein between the promoter and the therapeutic gene apositive-feedback system is provided, wherein said system is driven bythe promoter and controls by itself the expression of the therapeuticgene.
 15. The vector of claim 2, wherein between the promoter and thetherapeutic gene a positive-feedback system is provided, wherein saidsystem is driven by the promoter and controls by itself the expressionof the therapeutic gene.
 16. The vector of claim 10, wherein between thepromoter and the therapeutic gene a positive-feedback system isprovided, wherein said system is driven by the promoter and controls byitself the expression of the therapeutic gene.
 17. The vector of claim14, wherein the positive-feedback system comprises a T7 promoter and agene for T7 RNA polymerase.
 18. The vector of claim 15, wherein thepositive-feedback system comprises a T7 promoter and a gene for T7 RNApolymerase.
 19. The vector of claim 16, wherein the positive-feedbacksystem comprises a T7 promoter and a gene for T7 RNA polymerase.
 20. Thevector of claim 17, wherein the promoter controls the gene for T7 RNApolymerase and the T7 promoter controls the therapeutic gene, saidvector further comprising another expression unit comprising the T7 RNApolymerase under the control of the T7 promoter.
 21. The vector of claim18, wherein the promoter controls the gene for T7 RNA polymerase and theT7 promoter controls the therapeutic gene, said vector furthercomprising another expression unit comprising the T7 RNA polymeraseunder the control of the T7 promoter.
 22. The vector of claim 19,wherein the promoter controls the gene for T7 RNA polymerase and the T7promoter controls the therapeutic gene, said vector further comprisinganother expression unit comprising the T7 RNA polymerase under thecontrol of the T7 promoter.
 23. A retrovirus, coding for a vector ofclaim
 1. 24. A method for treating tumours by gene therapy, comprisingthe step of administering to an individual in need of such therapy avector of claim
 1. 25. The method of claim 24 for treating neoplasticdisorders.
 26. The method of claim 24, further comprising the step oftreating the individual with radiotherapy.
 27. The method of claim 24,further comprising the step of treating the individual with a therapywith cytostatics.
 28. A method for treating benign tumours, comprisingthe step of administering to an individual in need of such treatment avector comprising a gene under control of the cyclin-A promoter.
 29. Themethod of claim 28 for treating recurring stenoses.